LDHk is a novel isozyme of lactate dehydrogenase which we have identified in anaerobically shocked rat fibroblasts and in non-rat cells transformed by the Kirsten and Harvey murine sarcoma viruses. It is composed of 56,000 dalton subunits which readily cleave to 35,000 dalton and 22,000 dalton fragments. It is uniquely regulated, being reversibly inhibited by physiological concentrations of oxygen and by nucleoside triphosphates. It is readily isolated from and distinguished from other isozymes of LDH, although we have found it as a contaminant of commercial LDH-5. In biochemical studies of LDHk, we find that oxygen acts by increasing its Km for substrates by approximately 100-fold, while having no effect on its Vmax. We find that the 5',5'-dipurine nucleoside tetraphosphates Ap4A and Gp4G are exceptionally potent noncompetitive inhibitors of LDHk activity. Human tumors contain high levels of LDHk activity, ranging from 10-\to 500-fold higher than the levels in adjoining nontumor tissue. LDHk has been detected in the serum of over half of all patients examined (greater than 300), but is present in very few noncancer patients. Studies of LDHk expression in normal tissues have revealed that the retina also expresses high levels of this enzyme. This may relate to the long-known phenomenon that the retina shares with most tumors an unusual metabolism based on aerobic glycolysis.